Organellar ribonucleases and poly(A) polymerases in Chlamydomonas reinhardtii
RNA catabolism occurs in multiple cellular compartments. This study used the unicellular photosynthetic eukaryote Chlamydomonas reinhardtii to identify a small suite of putatively organellar, nucleus-encoded prokaryotic-like ribonucleases and poly(A) polymerases (PAPs) that may participate in this process. Other related genes encoded putative exosome components, but no nucleus or cytosol-specific exosome activating factors. A surprisingly large family of nine non-canonical PAPs were hypothesized to play a regulatory role in RNA degradation. The well-studied E. coli poly(A)-mediated RNA degradation pathway was the model used to predict and test the roles of candidate genes in chloroplast RNA catabolism. The pathway initiates with an endonucleolyic cleavage event, followed by polyadenylation of the resultant 3' end, which stimulates 5' to 3' exoribonucleolytic degradation. Unlike E. coli, Chlamydomonas has no RNase E and thus utilizes another enzyme, possibly RNase J1, as an endonuclease. Transient depletion of RNJ1 mRNA showed a very mild effect on chloroplast transcripts. As in E. coli, one hydrolytic (RNB2) and one phosphorolytic (Polynucleotide phosphorylase: PNPase) exoribonuclease may play a role in RNA decay. PNPase depletion caused an increase in RNB2 expression, but no profound alterations in the processing or steady state level of chloroplast RNA that would be expected if it were the sole exoribonuclease. Thus, it was concluded that there is likely to be some functional redundancy between these enzymes. PNPase depletion also revealed unique functional aspects, since it resulted in defects in acclimation to phosphate limitation, and was shown to be responsible for both polyadenylation and poly(A) tail degradation in the chloroplast. Comparative analysis of Arabidopsis and Chlamydomonas nucleotidyltransferases suggested that Chlamydomonas PAP4 but not PAP3 is likely to be mitochondrially localized. Recombinant PAP4 has polyadenylation activity, and when expressed in E. coli can partially complement a PAP1 (pcnB) mutant. Although PAP4 appeared to be mitochondrially targeted, its depletion affected neither mitochondrial poly(A) nor novel U-rich tails. Taken together, these studies have identified candidate genes encoding ribonucleases and poly(A) polymerases, elucidated some of the functions of PNPase in the chloroplast, characterized the biochemical activity of PAP4, and characterized polynucleotide tails in the mitochondria of Chlamydomonas reinhardtii.
Chlamydomonas; chloroplast; mitochondria; RNA degradation; ribonuclease; polyadenylation
Dissertation or Thesis